Previous proof-of-principle research will provide the foundation for the discovery and optimization of selective inhibitors of pyruvate kinase (PYK) of trypanosomatid parasites (Trypanosoma brucei, T. cruzi and Leishmania species). These pathogens cause serious, often fatal diseases of humans such as sleeping sickness, Chagas'disease and kala-azar in tropical and subtropical countries primarily in Africa, Central and South America, and Asia where many millions live in areas where the diseases are endemic. Tragically, current drugs for their treatment are unsatisfactory because they are toxic and ineffective against some forms of the diseases, and resistance is becoming increasingly common. Glycolysis is essential in T. brucei and therefore a promising drug target. Inhibitors of glycolytic enzymes such as PYK may thus serve as lead compounds for the development of new drugs. The proposed research has as specific aims: (1) to exploit unique features of trypanosomatid PYK (for which detailed structural information is already available and which has been validated as drug target by RNAi) for the discovery of selective inhibitors of PYKs from L. mexicana (LmPYK) and from T. brucei (TbPYK) through quantitative high-throughput screening of the Molecular Library Small Molecule Repository (MLSMR) containing 300,000 small molecules;(2) To confirm the potency of these compounds in a panel of secondary assays and to test the selectivity against human PYK, and to further improve the potency of the most promising molecules thus obtained by structure-based methods, analogue synthesis and medicinal chemical principles;(3) To test compounds displaying the highest potency for their ability to inhibit growth of cultured cells representing pathogenic stages of the parasites.